Field of the Invention
A process for determining the extent of environmental degradation of a human DNA sample by using newly identified target elements in a real time polymerase chain reaction system is disclosed.
Description of the Related Art
In the last recent years, real-time polymerase chain reaction (PCR) chemistry has become the standard for reliably quantifying the amount of genomic and amplifiable DNA in a forensic sample. Commonly used systems include the assessment of total human and male DNA. Examples are Quantifiler® from Life Technologies Corporation, Plexor® from Promega Corporation and Quantiplex® from Qiagen. Currently there are several different approaches used for fluorescence-based quantification assays, including SYBR® Green, Plexor®, TaqMan®, AmpliFluor®, Quantifiler® and Quantiplex®.
Interest has recently grown in using real-time PCR methods to evaluate the extent of degradation of a DNA sample. This may be done using two nuclear DNA targets: a short multi-copy sequence and a long multi-copy sequence. Because the long target sequence will degrade more rapidly than will the short target sequence as a sample is compromised, the ratio of the quantity of the short target to the long target will provide an assessment of the extent of degradation in the sample. Studies on the assessment of degraded DNA in a forensic sample have been published using Alu or mini-satellite targets. However, the assays of previous studies either lack in sensitivity or do not exhibit high PCR efficiencies. Forensic samples vary widely in quantity and quality, making the goal of developing and validating a real-time PCR system for the purposes of quantitating the DNA in these samples and determining the extent of their degradation a challenging one.
The recent advances in mini short tandem repeat (STR) analysis systems have now made it possible to analyze highly compromised samples. Investigators have made great strides in the development of STR amplicons that, compared with traditional STR amplicons, are reduced in size and can be used effectively on DNA samples that have been significantly degraded (see, e.g., J. M. Butler, et al., J. Forensic Sci. 48(5): 1054-1064 (2003); T. J. Parsons, et al., Forensic Science International: Genetics 1: 175-179 (2007)).
Alu are Short Interspersed Elements (SINE), approximately 300 bp insertions which are distributed throughout the human genome in large copy number. The evolution of Alu elements in the human genome over time has made Alu elements well suited for the task of distinguishing human DNA from non-human DNA and for doing testing that is desired to be specific to human DNA. A recent study reports an evaluation of the quality assessment of degraded DNA samples using a Ya5-lineage Alu genetic element (J. A. Nicklas, et al., J. Forensic Sci. 57(2): 466-471 (2012)). A multi-copy intra-Alu based approach for quantifying human specific DNA in an evidence sample has been successfully used to obtain DNA quantification with high sensitivity (J. A. Walker, et al., Anal. Biochem. 337: 89-97 (2005)).
The average age of Yb-lineage subfamily elements is estimated as 2.39 million years. It is estimated that the human genome contains over 1800 Alu Yb family elements and, out of those, approximately 50% are from the Yb8 subfamily. The Alu Yb8 system is known for the presence of a large number of fixed insertions. It has been reported that only 20% of the Yb-lineage Alu elements are polymorphic for insertion presence or absence in the human genome (A. B. Carter, et al., Human Genomics 1(3): 1-13 (2004)). Because a large number of these fixed elements are present in every human genome, the individual specific variation possible when using a multi-copy target quantification system is minimized.
In 1994, Shen, et al., identified a new composite retroposon when they studied the structure of the retinitis pigmentosa (RP) gene (Shen, et al., J. Biol. Chem. 269(11): 8466-8476 (1994)). This new retroposon consisted of the SINE-R element together with a stretch of sequence that shares sequence similarity with Alu sequences. Thus, it was named “SVA” after its main components, Short Interspersed Elements (SINE), Variable Number Tandem Repeats (VNTR) and Alu. SVA elements contain the hallmarks of retrotransposons, in that they are flanked by target site duplications (TSDs), terminate in a poly(A) tail and are occasionally truncated and inverted during their integration into the genome.